PROJECT SUMMARY Allogeneic hematopoietic stem cell transplantation (HSCT) plays a vital role in treating high-risk hematologic malignancies and some non-malignant conditions. However, many patients undergoing HSCT develop acute graft-versus-host disease (GVHD), and current prevention strategies that globally immunosuppress or selectively deplete T cells are associated with higher risks of cancer relapse, delayed immune reconstitution, increased infections, and other toxicities. Identifying the best way to suppress donor T cells to prevent GVHD while not losing their beneficial functions remains a critically unmet need. The adoptive transfer of murine invariant natural killer T (iNKT) cells significantly reduces GVHD, and a number of human studies support a role for iNKT cells in GVHD suppression. Several immunophenotypically and functionally distinct iNKT subsets have been defined in mice, and only certain subsets have the capacity to suppress GVHD. Despite these advances in understanding murine iNKT cells, the heterogeneity of human iNKT cells remains largely unexplored, and the subset best suited to prevent GVHD is unknown. The overall goal of this proposal is to define human iNKT cell heterogeneity and to develop strategies for the targeted selection and generation of immunosuppressive iNKT cells. To achieve this goal, similar approaches to those used in mice, as well as novel techniques, will be used to define the phenotypic, molecular, and functional characteristics of human iNKT subsets to delineate an immunosuppressive population with the potential to prevent GVHD. In addition, a gene-engineering strategy will be employed to enhance immunosuppressive function. These experiments will contribute substantially by providing the scientific basis for the isolation of immunoregulatory iNKT cells which will enable the design of a future clinical trial of iNKT cell adoptive transfer for GVHD prevention. Importantly, this work is broadly applicable beyond control of GVHD, as understanding human iNKT heterogeneity will provide a framework for future studies of iNKT cells and iNKT-based therapies in other disease states, including autoimmunity, cancer, and infection. The proposed project is part of a comprehensive career development plan for the applicant to build upon her substantial prior research experience in cellular immunology and hematopoietic stem cell transplantation. In particular, she plans to acquire new skillsets including high throughput proteomic and transcriptomic analyses and genetic engineering of cells. This training will occur under the guidance of her primary mentor, Dr. Robert Negrin, and her research advisory committee, including Drs. Maria Grazia Roncarolo, Matthew Porteus, Samuel Strober, and Holden Maecker. Their guidance and expertise, combined with that of her collaborators Drs. Ken Weinberg, Kara Davis, Nima Aghaeepour, and Everett Meyer, will ensure that the applicant completes the proposed studies and acquires the expertise necessary to fulfill her career goal of becoming an independent physician scientist with a research program focusing on immune regulation of GVHD in HSCT.